1. Technical Field
This application relates generally to field-flow fractionation. More specifically, the application provides apparatuses and methods for the discrimination of particles using sedimentation field-flow fractionation operating without the use of rotating-seal type coupling devices.
2. Prior Art
Field-flow fractionation (FFF) is a single-phase elution-based particle separation and characterization technique developed in the late 1960's by J. C. Giddings at the University of Utah. The first patents for the technique were issued to John C. Giddings in Jun. 17, 1969 (U.S. Pat. No. 3,449,938) and to Edward M Purcell and Howard C. Berg in Aug. 11, 1970 (U.S. Pat. No. 3,523,610). To effect a separation using FFF, the sample particles to be discriminated are introduced into a fluid medium that is passing through a narrow enclosed channel typically formed from two closely spaced parallel or concentric surfaces. The thickness of the channel, defined by the distance between the surfaces, is typically in the range of 25 to 300 micrometers and is much smaller than the other two dimensions of the channel. As the fluid medium moves through the channel, its flow rate is adjusted to achieve laminar flow conditions producing a differential flow profile that is parabolic or near-parabolic in shape. The rate of fluid flow is greatest in the middle of the channel but decreases progressively as the surfaces are approached.
To discriminate the sample particles in the fluid medium, one or more fields or forces are applied across the narrow thickness of the channel, perpendicular to the fluid flow. As the fields or forces interact with the sample, particles having different characteristics or properties aggregate into different steady state equilibrium zones across the parabolic fluid profile. The exact mechanism involved in establishing the breadth and position of the zones in the fluid profile depends on (1) the characteristics and properties of the sample particles in the fluid medium, (2) the nature of the applied field or force, (3) the strength of the interactive coupling between particular sample particles and the applied field or force, and (4) the extent to which the particular sample particles experience opposing secondary and/or dispersive interactions or forces in the channel. Particles that aggregate into zones in the more rapidly moving fluid exit from the channel first. Other zones then follow depending on their relative position in the flow profile. Because of the differential nature of the fluid flow, sample particles elute from the channel at different times, thus providing discrimination and separation.
Since the inception of FFF, applications have continued to grow in number encompassing such diverse areas as biomedical research, environmental studies, industrial colloids, natural and synthetic polymers, mining, and pharmaceuticals. Being an elution technique, FFF is readily adapted to fraction collection and online coupling to almost any liquid chromatographic or particle detection system. Sample particles can range in size from 0.001 to 100 micrometers with little restriction on their form or composition. The versatility of FFF comes from the ability to tailor the type and strength of the field or force to the specific properties of the particles to be separated. It was even recognized fairly early in the development of FFF (summarized in Giddings, 1993) that living cells could be separated without loss of viability or functionality.
Of particular interest in the present work is sedimentation field-flow fractionation (SdFFF). In this mode, the force applied across the channel is sedimentary in nature and generated by rotating the channel around an axis at an appropriate speed. Because of the way the force is created, SdFFF is also sometimes called centrifugal field-flow fractionation. To avoid confusion, it should be pointed out that SdFFF as defined above is distinctly different from gravitational field-flow fractionation (GFFF) where the sedimentary force is due to gravity. The acceleration of gravity can be taken as a fixed value. In SdFFF, the force depends on the radial distance of the channel from the axis and the angular velocity of the rotation. The force can therefore be adjusted to a desired value or programmed to change with time and is independent of the orientation of the channel relative to the earth.
SdFFF is one of the more uniformly applicable modes of FFF and probably the most selective, being able to discriminate particles differing by as little as 5-10% in size. Particles are separated based on the universal properties of mass and volume. As most review articles about SdFFF point out (Giddings, 1993), however, the technique also has shortcomings, principal among them being the complexity of the instrumentation. Unlike in other modes of FFF where the channel is stationary and generally linear, in SdFFF the channel is curved and must be rotated about an axis to generate the required force. The difficulty generally arises in that a means must be provided to maintain fluid communication between the rotating channel and stationary components within the instrument.
The present state of the art for SdFFF instrumentation is typified by the devices described in U.S. Pat. Nos. 4,283,276 and 7,442,315 where the sedimentation force is generated by attaching the channel to the rotor of a centrifuge. Fluid communication is made to the channel using rotating-seal type coupling devices (or more simply called rotating seals). Typically, to provide a continuous supply of fluid medium to produce the laminar flow required in the channel, fluid is pumped through narrow bore tubing from a reservoir of some type to the channel. The particles to be separated are added to the fluid using an injection device usually placed between the pump and the channel. After passing through the channel, the fluid is directed through additional tubing to a detection device that may be used to monitor and/or characterize the sample particles being separated. To keep the tubing from twisting and becoming tangled as the channel rotates, the rotating seals are used in connecting the tubing between the rotating channel and the stationary pump (or injection device) and detection device.
Although simple in concept, it is the rotating seals that create most of the problems and limitations in the use of SdFFF. The principal troubles come from leakage, contamination, and sample damage, particularly when the seal is operated at high rotational speeds. In its most basic form, the seal is constructed of two cylindrical members, one rotatable and the other non-rotatable, in face-to-face contact with a small diameter hole bored straight through the center of both members and the interface between. The hole aligns along the rotatable axis of the rotatable member. To minimize voids that can contribute a loss of sample separation resolution, the size of the hole is generally kept below 0.02 inches (0.5 mm).
Leakage, which is typically the greatest problem with rotating seals, is generally due to wear at the contact interface as the rotating members slide against one another. Additional leakage is also caused by misalignment and separation between the cylindrical members brought about by vibration as the members rotate. Heat due to friction can further exacerbate the wear and can ultimately limit the rotational speed at which the seal can be operated. Heat and shear forces at the interface can also damage fragile sample particles. In addition, debris from wear can restrict flow and contaminate fluid as it passes through the seal interface. Numerous efforts have been made in prior art to minimize the effects of these problems as illustrated by U.S. Pat. No. 4,502,699 and the other patents cited therein. Lubricants and cooling systems have been introduced to control friction and the heat that results. Tension springs, cushion mounts, and flexible connections have been incorporated into the designs to help diminish vibration as the seal rotates. Although effective to varying degrees, most of the improvements add complexity and additional cost to the seal design while still not totally eliminating the problems. The rotating seals remain high maintenance and are the weak link in the SdFFF apparatus.
Another difficulty created by the use of rotating seals is that the seals limit the number of rotating lines of communication that can be established with the SdFFF channel and thus severely restrict the ways the SdFFF apparatus can be configured and operated. Since rotating seals must be mounted along the axis of rotation for the channel, the single-bore rotating seal described above can provide the SdFFF apparatus with only two lines of fluid communication, one on either side of the channel. The use of such seals therefore precludes expansion of the SdFFF apparatus to more sophisticated higher-order techniques employing multiple inlet/outlet ports, multiple independent SdFFF channels and/or the use of multiple fields or forces where additional rotatable lines of communication to the channel would be required.
As evidenced by the absences of examples in the literature, even the use of a concentric double-bore rotating seal as described in U.S. Pat. No. 4,502,699 provides little improvement over the use of the single-bore models in the development of higher-order techniques in SdFFF. The use of a plurality of channels, for example, could save considerable operator time and effort by enabling multiple samples or calibration standards to be analyzed or “screened” simultaneously under a variety of experimental conditions. This is particularly relevant in clinical and commercial applications where throughput is often an important factor. These capabilities could even be further expanded by using multiple fields and ports to provide greater resolution through two-dimensional operation and more selective particle isolation.
The rotating-seal type coupling devices currently employed in SdFFF are a major source of problems in both reliability and maintenance. In addition, the seals dramatically limit the ability to expand the instrumentation to permit more advanced, higher-order separation capabilities. It would therefore be of substantial interest and benefit to develop apparatuses with associated methodologies that would enable the SdFFF discrimination of sample particles without the use of rotating-seal type coupling devices to provide fluid communication to the SdFFF separation channel.
Accordingly, a need remains for an apparatus and method for sedimentation field-flow fractionation in order to overcome the above-noted shortcomings. The present invention satisfies such a need by providing an apparatus and method for sedimentation field-flow fractionation that is versatile in its applications, and overcomes one or more of the above-noted shortcomings.